The long-term objective of this training award is to develop an independent research career with an emphasis on understanding the relationship between host-derived reactive nitrogen species and the disease-causing potential of Campylobacter jejuni. This Gram-negative organism is the most common cause of food-borne bacterial diarrheal disease in the United States. Despite this large burden of disease, little is known about the pathogenesis of C. jejuni compared to Enterobacteriaceae such as Salmonella. One antimicrobial host response is the production of toxic reactive nitrogen species (RNS) via inducible nitric oxide synthase (iNOS), but the antimicrobial effects of RNS on C. jejuni are not well-studied. Our preliminary results show that C. jejuni is more sensitive in vitro to nitrosating agents compared to Salmonella typhimurium. In a gentamicin-protection invasion assay with human colonic epithelial cells and a chemical iNOS inhibitor, we found about a 50% increase in the recovery of wild-type and acapsular C. jejuni but not S. typhimurium, suggesting that C. jejuni also is particularly susceptible to RNS in a host cell setting. The role of the C. jejuni capsule in protection from RNS is currently unclear. To extend this work, we will further explore the interaction of RNS with C. jejuni and the role of capsule in our invasion assay. To identify genes involved in resistance to nitrosative stress, we will perform random transposon mutagenesis in concert with measurement of susceptibility to nitrosating agents. To assess the biological significance of these genes, we will measure the ability of peritoneal macrophages from iNOS-null mice to kill RNS-hypersusceptible strains versus the wild-type. Finally, iNOS-null mice will provide an opportunity to measure colonization and invasion of wild-type and RNS-hypersusceptible strains in an oral infection model. The proposed studies will lead to a greater understanding of the pathogenesis of C. jejuni. The co-sponsorship of this award provides a rich environment in which the trainee will gain the technical and professional development needed to become an independent investigator.